Uridine (U) insertion/deletion RNA editing in kinetoplastid parasites is a remarkable posttranscriptional remodeling process whereby mitochondrial mRNAs undergo precise insertion/deletion of hundreds/tens of U residues to create translatable mRNAs. Edited sequence is specified by trans-acting guide RNAs (gRNAs), and dozens of gRNAs act sequentially in a 3' to 5' manner to facilitate complete editing of most mRNAs. The holoenzyme that mediates kinetoplastid RNA (kRNA) editing comprises the catalytic RECC (RNA Editing Core Complex) and non-catalytic RESC (RNA Editing Substrate Binding Complex) subcomplexes. RECC and RESC interact transiently during the editing process, and RESC is comprised of at least two modules whose interactions also appear dynamic. Published and preliminary data demonstrate that the DEAD box RNA helicase, REH1 (RNA Editing Helicase 1), also transiently associates with the kRNA editing holoenzyme, and REH1 is required for editing of a subset of mRNAs. Although one study linked REH1 to the 3' to 5' progression of editing, and to the unwinding of one mRNA-gRNA pair, the scope and mechanism of action of REH1 in kRNA editing are very poorly understood. RNA helicases have wide-ranging functions including unwinding RNA duplexes, facilitating RNA annealing, removing proteins from RNA molecules, and even stabilizing large ribonucleoprotein (RNP) complexes. Here, we propose two Aims towards determining the role(s) of REH1 in kRNA editing. In Aim 1, we will use an innovative bioinformatic tool developed in our laboratory to define the transcriptome-wide impact of REH1 depletion on kRNA editing progression. By analyzing partially edited mRNAs that accumulate in the absence of REH1, we will determine whether REH1 impacts gRNA exchange or gRNA utilization, and define the positions of these defects along the entire set of mitochondrial mRNAs. These analyses, along with evaluation of mis-edited junction sequences that accumulate upon REH1 knockdown, will provide insights into REH1 function at a previously unattainable level. We will then use mutational studies to elucidate the impact of REH1 ATP binding on its editing functions. In Aim 2, we will define the role of REH1 in remodeling the kRNA editing holoenzyme. Using RNA immunoprecipitations and co-immunoprecipitations with our extensive antibody panel, we will determine whether REH1 knockdown affects RECC-RESC dynamics and/or intra-RESC dynamics and RNA trafficking. The proposed studies will address important gaps in our knowledge of the kRNA editing mechanism, providing significant insights into a gene regulatory process that is essential for parasite viability and virulence. Additionally, they will expand our understanding of how DEAD box RNA helicases impact the dynamics of complex RNPs. !